B. burgdorferi survives and proliferates in both an arthropod vector and various mammalian hosts. During transmission, it encounters environmental challenges specific to those hosts. One such challenge comes from reactive oxygen species (ROS) e.g. superoxide radicals (O2.-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH.) and reactive nitrogen species (RNS) e.g. nitric oxide (NO), N2O3 and peroxynitrite. There are different stages in the infective cycle (in feeding ticks) when B. burgdorferi is exposed to ROS/RNS. One ROS/RNS challenge occurs as the bacteria migrate through the salivary glands during transmission. Our lab has demonstrated that the salivary glands of Ixodes scapularis contain significant levels of ROS and RNS. ROS are generated as a result of incomplete reduction of oxygen by salivary gland cells during normal metabolism. RNS are generated only during feeding when the salivary cells produce NO via an inducible NO synthesase (iNOS). NO has multiple biological effects on the host tissues, the most important of which is vasodilation. Surprisingly, midguts from feeding ticks also contained detectable levels of ROS and RNS. These data suggest that B. burgdorferi, during feeding, encounters ROS and RNS in the midgut and during passage through the salivary glands. Therefore, our current working model is that before B. burgdorferi migrates from the microaerobic midgut (containing ROS and RNS) to the salivary glands, ROS or RNS act as a signal(s) to induce the expression of ROS and RNS defense enzymes and key virulence factors that promote the survival and successful colonization of a new host. Cellular defenses against the damaging effects of ROS involve both enzymatic and nonenzymatic components. B. burgdorferi has a limited number of enzymes that could potentially be involved in this defense response. Those identified include a Mn-dependent superoxide dismutase (SOD), a Dps/Dpr homologue (NapA), thioredoxin (Trx), thioredoxin reductase (TrxR) and a Coenzyme-A disulfide reductase (CoADR). To date the Mn-SOD, CoADR, and NapA have been characterized experimentally. Because these enzymes would promote the in vivo survival of B. burgdorferi cells when challenged by O2.- and H2O2 from host cells, we are particularly interested in the process and how these molecules affect gene regulation that promotes transmission and survival. The Borrelia oxidative stress regulator, BosR, acts as a transcriptional activator of oxidative stress genes. Although there is no apparent amino acid homology (>15%), BosR appears to be functionally similar to OxyR from E. coli. In addition, we and other research groups have recently shown that BosR not only regulates sodA , NapA (a lipoperoxidase) and CoADR but also regulates the genes encoding key virulence factors (e.g., Outer surface protein C, OspC). This regulatory effect seems to be directed by BosR through the RrP2-RpoN-RpoS regulatory cascade. It seems clear that BosR- and RpoS-dependent regulation are critical during the transmission of B. burgdorferi. The effects of ROS/RNS on cells have been extensively investigated. These highly reactive compounds have been shown to damage cellular macromolecules including DNA, proteins, and membrane lipids. In eukaryotes, membrane lipids are a major target of reactive oxygen species. The most damaging effects of ROS in bacteria result from the interactions of radicals (H2O2) with free Fe2+ generating very reactive OH- (Fenton reaction). Because of this reactivity, its effect on any given biomolecule will depend largely upon proximity to the target. Because Fe2+ localizes along the phosphodiester backbone of nucleic acid, DNA is a major target of OH-. This reactive species can pull electrons from either the base and sugar moieties producing a variety of lesions including single and double strand breaks in the backbone and chemical crosslinks to other molecules. These strand breaks, and other lesions that block DNA replication, contribute to OH.- toxicity and cell death. The intracellular biochemistry of B. burgdorferi suggests that the primary intracellular target of ROS may not be DNA as described in other bacteria. In E.coli, the extent of DNA damage due to H2O2 and Fenton chemistry is directly proportional to Fe metabolism and the free Fe concentration within the cell (5-100 nM). Since the intracellular Fe concentrations of B. burgdorferi are estimated to be <10 atoms per cell, it seems unlikely that DNA is a primary target for ROS in B. burgdorferi. In support of this, B. burgdorferi cells exposed to toxic levels of ROS had no measureable DNA damage. This suggested that lethality of ROS in B. burgdorferi was due to damage of other cellular components. As previously mentioned, B. burgdorferi incorporates polyunsaturated fatty acids from the environment into the cells membrane lipids and lipoproteins suggesting that Borrelia membranes could be a target for lipid peroxidation. Analyses of t-butyl peroxide treated B. burgdorferi cells by electron microscopy showed significant irregularities indicative of membrane damage. Fatty acid analysis of cells treated with t-butyl peroxide and lipoxidase indicated that host-derived linoleic acid had been dramatically reduced (10- and 50-fold, respectively) in these cells, with a subsequent increase in the levels of malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) aldehyde(4- and 10-fold respectively), the toxic by-products of lipid peroxidation. These data, taken together, suggest that, in wild-type B. burgdorferi membrane lipids and lipoproteins are the primary targets for attack by ROS. These data suggested that no DNA damage occurs in B. burgdorferi challenged with ROS. As previously stated, the lack of DNA damage was attributed to the lack of intracellular Fe and thus, no DNA oxidizing Fenton chemistry. However, mutations in genes encoding proteins required for base excision repair (BER), mismatch repair (MMR) or nucleotide excision repair (NER) have a dramatic effect on susceptibility of cellular DNA to oxidative damage. This damage appears to be the result of Cu-mediated Fenton chemistry which has never been observed in other living systems. While lipids would still be considered to be the primary target of ROS in wild-type B. burgdorferi, DNA damage can occur but lesions are repaired successfully by the BER, NER and MMR systems. While B. burgdorferi is resistant to killing by reactive oxygen species (ROS), yet, previous studies have demonstrated that it is highly susceptible to killing by reactive nitrogen species (RNS). In this study, diethylamine NONOate (DEA/NO) was used to characterize the lethal effects of RNS on B. burgdorferi. RNS produce a variety of lethal DNA lesions, however, levels of the DNA deamination product, deoxyinosine, as well as the number of apurinic/apyrimidinic sites were identical in DNA isolated from untreated and DEA/NO-treated cells. The lack of DNA damage in DEA/NO-treated B. burgdorferi was due, in part to the activity of the Nucleotide Excision Repair (NER) pathway as shown by hypersensitivity of uvrC- and uvrB-deficient strains of B. burgdorferi to RNS, along with an increased spontaneous mutation rate leading to coumermycin A1 resistance. Polyunsaturated fatty acids in B. burgdorferi cell membranes, which are susceptible to peroxidation by ROS, were not sensitive to RNS-mediated lipid peroxidation (assayed by measuring malondialdehyde). However, proteins from DEA/NO-treated B. burgdorferi cells displayed a high degree of S-nitrosylation, as well as increased levels of cytoplasmic zinc (measured with Zinquin) indicative of damage to free and zinc-bound cysteine thiols (e.g., BosR, NapA and fructose-1,6biphoahphate aldolase). These data suggested that proteins with free or zinc-bound cysteine thiols are the major targets of RNS in B. burgdorferi.